5I 5
J. gen. ViroL (I973), 2I, 515-52I
Printed in Great Britain
The Replication of Polyoma D N A
By L. V. C R A W F O R D , C H R I S T I N E S Y R E T T AND A N N E L I E W I L D E
Imperial Cancer Research Fund Laboratories, P.O. Box 123,
Lincoln's Inn Fields, London, WC2A 3P,Y
(Accepted 3I July I973)
SUMMARY
Measurement of replicating molecules of polyoma virus DNA after digestion
with endonuclease R1 shows that DNA replication is bidirectional, starting predominantly at a specific site. In both large plaque and small plaque polyoma virus
DNA this site is 29 _ 2 ~ of the total length of the DNA from the cleavage site of
the endonuclease R1.
INTRODUCTION
It has been established that polyoma virus DNA and Simian virus 4o (SV 40) DNA
replicate by the mechanism first proposed by Cairns (1963) and Hirt (1969). Further studies
(Bourgaux & Bourgaux-Ramoisy, 1971, I972) have elucidated some of the properties of the
intermediates in replication. The replication of SV 40 DNA has been shown to start at a
specific site, proceed bidirectionally, and terminate at a specific site (Danna & Nathans,
I972; Fareed, Garon & Salzman, I972; Nathans & Danna, I972). It is of interest to know
whether the replication of polyoma virus DNA follows the same pattern.
METHODS
Virus stocks. One stock of small plaque (Crawford, I962) and one stock of large plaque
(Dulbecco, Hartwell & Vogt, i965) , polyoma virus were used for this study. Both stocks
had been recently plaque purified and grown at low multiplicity of infection. The quality
of the virus was checked by determining that the DNA produced in one cycle of growth at
high multiplicity was more than 9o% sensitive to the restriction endonuclease R1.
Isolation of replicating DNA. Mouse embryo fibroblast secondary cells were infected at
an input multiplicity of IO to 2o p.f.u./cell and grown for 24 or 30 h at 37 °C in Dulbecco's
enriched Eagle's medium (E 4), with IO ~ calf serum. Medium was removed from the
cultures and the DNA extracted with sodium dodecyl sulphate by the method of Hirt
0967). The small DNA in the Hirt supernatant fraction was phenol extracted, precipitated
with ethanol and centrifuged in ethidium bromide/CsC1 gradients (200/zg/ml ethidium
bromide; density 1"58 g/ml) in a 5oTi angle rotor (Beckman L2 65B centrifuge), at
40ooo rev/min for 48 h. After centrifuging the tubes were examined in u.v. light to locate
the lower (covalently closed circular DNA) and upper (linear and relaxed circular DNA)
bands. These two bands, and the region between them, were collected separately.
The ethidium was removed from the samples by repeated extraction with isopropanol,
saturated with 50 ~ (w/w) CsC1, and the samples dialysed against tris buffer (O'OI M,
pH 7"5) containing mM-EDTA. In some cases, the DNA was concentrated by precipitation
with two voI. of ethanol and the precipitate resuspended in the same buffer.
R1 endonuelease cleavage. The R1 endonuclease was isolated by Dr W. R. Folk, following
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L.V. CRAWFORD,
C. S Y R E T T A N D A. W I L D E
the procedure of Yoshimori 097~) from Escherichia coli RY-~3, an endonuclease I- strain
carrying the resistance transfer factor R 1. DNA was digested in tris-buffered (o'05 M,
pH 7"5) saline (o.x M-NaC1) containing MgCI~ (o.o~ M). Digestions were carried out at
37 °C usually for I5 min, with the amount of enzyme adjusted to cleave 5o to 9o ~ of the
polyoma DNA molecules, as determined by preliminary experiments. Since the R1 endonuclease may not be completely specific nor completely free from other less specific endonucleases it was considered desirable to avoid treating the DNA with an excess of enzyme,
since this might cause cleavage of the replicating molecules at sites other than the R1 site.
Most DNA preparations contain defective molecules which are resistant to digestion by the
R~ endonuclease. Cleavage of such molecules by endonucleases other than R1 would be
expected to give rise to molecules in which the apparent origin of replication was not in its
normal position.
Electron microscopy. Samples were prepared for electron microscopy by the basic protein
film technique of Kleinschmidt, using the aqueous method of Davis, Simon & Davidson
(t97Q. The DNA sample in 0"5 M-ammonium acetate with Ioo #g/ml cytochrome C was
layered on to a hypophase of o.25 M-ammonium acetate. The film was picked up on parlodion-coated copper grids, stained with Io -8 M-uranyl acetate in 90 ~ ethanol, dried with
ethanol and rotary shadowed with platinum:palladium (8o:2o). Micrographs were taken
with a Siemens ~oI at an instrumental magnification of × ~oooo, and the DNA molecules
measured at a final magnification of × 90oo0.
RESULTS
The techniques used here were based on those developed for SV 40 by Fareed et al.
(I972). Replicative intermediates were isolated by centrifuging to equilibrium in CsC1
gradients containing ethidium bromide and then cleaved at a specific point with the restriction endonuclease R 1 (Yoshimori, ~97I). This enzyme cuts DNA at a specific sequence,
which has been determined (Hedgpeth, Goodman & Boyer, ~972) and, as this occurs only
once in SV 4o DNA (Morrow & Berg, 1972), converts circular DNA into linear DNA of
the same length. The same is true for polyoma virus. Polyoma DNA is cleaved by endonuclease R1 to full length linear molecules and these give rise to linear molecules, not
circles, when denatured and reannealed, showing that they were all cut at the same point
(D. Robberson, personal communication). The results reported here confirm this, since
the distance of the origin of replication from the cleavage site would be constant (see below)
only if both the following conditions were satisfied. Firstly, that the endonuclease R1 always
cleaves the replicating molecules at the same site and, secondly, that the origin of replication
is at a specific site on the DNA. The R1 site forms a convenient reference point on the
DNA and the positions of the replication forks in the replicative intermediates can then be
determined relative to this site.
In CsC1 gradients containing ethidium bromide, replicating circular duplex DNA has a
buoyant density between that of non-replicating covalently closed circular DNA (~.59 g/ml),
and that of open circular DNA (I.55 g/ml). DNA, extracted from cells infected with small
plaque polyoma virus, was centrifuged to equilibrium in a CsCl-ethidium bromide gradient.
The interband DNA, when examined by electron microscopy, contained many replicating
molecules, although it was contaminated with supercoiled DNA from the lower band and
linear plus open circular DNA from the upper band.
Before R1 endonuclease digestion, many of the replicating molecules were too tightly
supercoiled for the entire contour length of the DNA to be discerned. This was previously
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Replication of polyoma DNA
517
Fig. i. Replicating molecules of small plaque polyoma virus DNA cleaved by endonuclease R~.
Replicating polyoma virus DNA preparations were digested with endonuclease Ra and prepared
for electron microscopy by the aqueous method.
observed in the replication of SV 40 by Sebring et aL 097~). After R 1 cleavage, the molecules assume the linear configuration shown in Fig. I with a replication loop, a long arm
and a short arm. Since R1 endonuclease cleaves polyoma virus D N A at a specific site, these
forms must have been derived from Cairns type circles, all of which have been opened at
the same site. Cleavage at this site then generates the two free ends of each molecule.
Measurements were made on molecules of the type shown in Fig. I, with the results
summarized in Fig. 2. As the replicated region becomes larger, both arms (LI and L4)
become shorter, i.e. the replication forks approach the R1 site. After replication passes
through the R~ site, the appearance of the molecule changes to that of a double Y (Fig. 3)
The distance of the origin of D N A replication from the R 1 cleavage site was estimated
as [¼(L2 + L 3)] + L I for the set of molecules measured for Fig. 2. For small plaque polyoma
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L.V. CRAWFORD~ C. SYRETT AND A. WILDE
60
i
i
I
L2
L4
50
L3
40
t
30
-%
00
":o \°
-o~
o°
IQ
0
20
10
I
0
10
20
L 1 (~ total length)
30
40
Fig. 2. The relationship of the extent of replication and the position of the replication loop in
endonuclease R1 cleaved small plaque polyoma virus DNA. Molecules of the type shown in Fig. I
were measured and their dimensions are shown in this figure, The extent of replication ½(L2 + L 3),
expressed as a percentage of the total length (L I + [½(Lz + L 3)] + L 4), has been plotted against L I,
also as a percentage of the total length.
virus this origin occurs at a position corresponding to 29 + 2 ~oo from the R1 site, taking the
length of the molecule (L[ + ½ ( L 2 + L 3 ) + L 4 ) as IOO ~ . The actual value obtained was
28.67 ~ and since the standard deviation was _+2 ~ this has been rounded off to 29 ~ .
This is the origin of the line shown on Fig. 2, extrapolating to L I equals 29 ~ at zero
replication and to L I equals zero at 58 ~ replication. All the points in Fig. 2 fall close to
the line as expected for R1 endonuclease cleavage at a unique site, when the replication is
bidirectional from a unique origin with both replication forks moving at similar rates. The
point at which replication terminates is not localized precisely but it is likely to be the
opposite side of the circle from the origin, i.e. 79 ~ of the molecule from the R~ site. The
small number of double Y molecules observed are consistent with this interpretation. Some
molecules, similar in general appearance to those shown in Fig. ], have not been included
in Fig. 2 or subsequent calculations. When the two sides of the replication loop were of
unequal length (by more than 2 ~ of the overall length of the molecule) (about 20 ~ of the
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Replication of polyoma DNA
519
Fig. 3. Replicating molecules of small plaque polyoma virus DNA. These molecules are from the
s~me preparation as shown in Fig. ~ but differ from them in that replication has extended beyond
the R1 cleavage site.
60
eo
I
I
I
L4
50
40
3O
-..:\::
•
•
0 0
20
•
~ 0T M
•
•
•
•
•
Q
10
I
0
10
20
L I (",i total lenglh)
30
40
Fig. 4. The relationship of the extent of replication and the position of the replication loop in
endonuclease R1 cleaved large plaque polyoma virus DNA. Replicating D N A molecules were
isolated from cells infected with large plaque polyoma virus and treated with endonuclease R1.
The relative length of the short arm L I has been plotted against the extent of replication, as for
Fig. 2.
34
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520
L . V . C R A W F O R D , C. SYRETT AND A. W I L D E
molecules), or when the length of the molecule was less than that of the open circular or
linear polyoma D N A measured on the same grids (about IO ~ of the molecules), the
molecule was not included in the analysis. Most of this variation is probably due to uneven
spreading of the molecules during preparation for electron microscopy. Some of the
smaller molecules may be defective DNAs in which parts of the virus D N A are missing or
have been rearranged.
Similar experiments with large plaque polyoma virus gave the results shown in Fig. 4.
Replication appears to follow the same course as seen with small plaque virus and the origin
of replication is located at 29"3 _+2 ~oo from the R~ site. This is, therefore, not significantly
different from the value obtained for small plaque polyoma virus.
DISCUSSION
The D N A replication of the two strains of polyoma virus studied appears to be similar in
several respects to that of SV 4o D N A (Danna & Nathans, I972; Fareed et al. I972;
Nathans & Danna, I972 ).
The replicating polyoma virus D N A molecules, after R1 digestion, can be arranged in
an orderly series, indicating a unique origin for the bidirectional replication, assuming that
the R1 endonuclease cleaves at the corresponding site in each molecule or at a small number
of sites very close to each other. The average overall length of all the replicating molecules
observed was not significantly different from that of non-replicating open circular DNA.
Thus no detectable length of D N A was removed by R~ treatment.
The symmetry of this array of replicative forms indicates that replication proceeds in
both directions from a unique origin at comparable rates. Unidirectional replication would
give rise to molecules in which either L I or L 4 would remain constant as replication
proceeded, and this is clearly not the case here.
The positions determined for the origin of replication, at 29 ~ of the genome from the
R1 site, are similar for large plaque and small plaque polyoma virus. The origin for SV 4o
D N A replication occurs at 33 ~ from the R1 site (Fareed et al. 1972 ). Since the virus DNAs
are circular there is an ambiguity in positioning the origin of replication by these studies
alone. The origin located at 29 ~o of the genome from the R~ site may in fact be positioned
at 7I ~ 0 o o - 2 9 ) from the Rx site as measured in the opposite direction. To resolve this
ambiguity it is necessary to locate some other marker on the D N A and experiments are
now in progress to locate the attachment site for phage T 4 gene 32 protein (Delius, Mantell
& Alberts, 1972) on replicating molecules relative to the Rx site and the origin of replication.
This should allow an unambiguous positioning of the replication origin in the two strains
of polyoma virus DNA. Preliminary experiments indicate that the gene 32 protein attachment site in polyoma virus D N A extends from approximately 2o to 25 ~ of the total length
of the D N A from the R1 cleavage site (M. Yaniv, O. Croissant & F. Cuzin, personal
communication; and our unpublished results with Dr J. Monjardino). This is true for both
large plaque and small plaque virus. In R1 cleaved replicating molecules gene 32 bubbles
have not been seen in the long arm, L4 of the molecules. This indicates that, measuring
in the same direction from the R1 site, the gene 32 site is likely to be at 2o to 25 ~ and the
origin of replication at 29 ~.
The comparison of polyoma virus DNA with SV 4o DNA is more difficult since it is not
known whether the R~ site is located in a region with equivalent biological function in the
two viruses. With the accumulation of additional positional markers on polyoma virus
D N A it should be possible to make more meaningful comparisons for the functional
anatomies of the two virus DNAs.
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Replication of polyoma DNA
521
We would like to thank Dr W. R. Folk for the preparation of R1 endonuclease used,
Dr D. L. Robberson for his helpful comments and Alan Robbins and Kit Osborne for
their assistance with preparation of virus stocks and DNA samples.
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